Root extracts of Cryptolepis decidua are used in the traditional medicine of South-West Africa to treat various conditions. Here, we report the potent cytotoxic activity of a methanol root extract against a human adenocarcinomic cell line, which prompted us to establish a comprehensive metabolite profile. Overall, six previously undescribed compounds were isolated from this extract by a combination of open-column chromatography (CC), centrifugal partition chromatography (CPC), and preparative HPLC. They include two sarmentogenin-type cardenolides (2 and 3), an androstane lactone (5), two dinormonoterpenoid diglucosides, dinosides A and B (9 and 10), and a quinic acid derivative (12). In addition, six previously reported constituents were identified (1, 4, 6-8, 11). The structures of the isolates were elucidated using HPLC-MS2/3, HRESIMS, and NMR spectroscopy. Feature-based molecular networking was used to map a comprehensive metabolite profile. The cytotoxic activity of the methanol extract was evaluated by WST-1 cell-viability assays, apoptosis/necrosis tests, and cell-cycle analyses. Cardenolides 1-3 were identified as the primary cytotoxic agents, exhibiting IC50 values of 0.25 μM (1), 2.20 μM (2), and 0.28 μM (3) in A549 human lung epithelial cancer cells, and 0.41 μM (1), 2.31 μM (2), and 0.39 μM (3) against peripheral blood mononuclear cells (PBMCs).

Genes involved in the biosynthesis of microbial natural products (NPs) are typically arranged in biosynthetic gene clusters (BGCs). Different congeners of an NP family typically possess distinct chemical features introduced by additional tailoring enzymes encoded in the corresponding BGC variants. However, tools to rapidly visualize the core gene set and distinguish it from variant-specific tailoring genes (VSTGs) in these BGCs are lacking. Here, the software tool BisCEET (Biosynthetic Cluster Environment Examination Tool) was developed, allowing comparison and visualization of the gene composition of related BGCs, thereby streamlining the identification of VSTGs in uncharacterized BGC variants and strains likely to produce novel NP congeners. The use of BisCEET is exemplified by analyzing bacterial BGCs of staurosporine-like indolocarbazoles and xantholipin-like polyketides, which enabled the identification of numerous apparent BGC variants. We anticipate that BisCEET will become a valuable bioinformatic asset, streamlining the prioritization of BGCs and the cultivation of microbial strains for the discovery of distinct NP variants and novel tailoring enzymes.

Aromatic polyketides from Actinobacteria are structurally complex bioactive natural products with significant therapeutic potential, whose biosynthesis involves polyketide chain assembly, keto reduction, cyclization, and aromatization. This is followed by pathway-specific enzymatic tailoring steps, occasionally including rare oxidative rearrangements of the carbon skeleton, as exemplified by the rishirilides. In this study, we investigate RslO9, a flavin-dependent tailoring key enzyme of rishirilide biosynthesis, previously hypothesized to facilitate a lactone-forming Baeyer-Villiger oxidation of the rishirilide naphthoquinone core and subsequent intramolecular aldol condensation. Through detailed investigation of RslO9's mechanism, structural features, and substrate scope, we unexpectedly found that the naphthoquinone moiety of the non-natural substrate lapachol undergoes hydroxylation followed by a benzilic acid rearrangement, producing the Hooker intermediate-a hallmark of the intricate Hooker oxidation. Our data support a similar alkyl migration mechanism for RslO9's native substrate, upending its prior classification as a Baeyer-Villiger monooxygenase and challenging the proposed role of related enzymes while also providing a novel framework for exploring their catalytic roles.

Aromatic polyketides from Actinobacteria are a class of natural products with potential for clinical application due to their antibiotic and cytotoxic activities. Among the most complex members are the closely related rubromycins and griseorhodins, which are derived from a pentangular backbone that undergoes extensive oxidative tailoring to afford a characteristic bisbenzannulated spiroketal pharmacophore. Recent insights into the characteristic genetics and enzymology underlying the biosynthesis of these polyketides now enable the bioinformatics-driven search for further congeners. Here, we analyzed 154 available and manually selected rubromycin/griseorhodin biosynthetic gene clusters (BGCs) with the help of the tool BisCEET, which allowed the rapid identification and visualization of "variant-specific tailoring genes" (VSTGs), encoding putative tailoring enzymes required only for individual congeners. This approach revealed a striking BGC variant in the genome of the Actinomycete Actinacidiphila soli, predicted to enable the biosynthesis of a glycosylated griseorhodin variant. Following the successful cultivation of the strain, the production of a previously uncharacterized griseorhodin, named ruskamycin, was confirmed that featured a distinctive digitoxose-substituted epoxy-spiroketal pharmacophore and exhibited antibacterial activity against Gram-positive strains.

The identification of antiplasmodial lead compounds from the aerial parts of Vernonia glabra (Asteraceae) was carried out using a bioassay-directed protocol. The methanolic extract of the plant was partitioned into four main fractions using n-hexane, dichloromethane, ethyl acetate, and n-butanol. These fractions were then tested for their antiplasmodial activity against Plasmodium falciparum 3D7. The bioassay results indicated that the dichloromethane-soluble fraction had the highest potency, with an IC50 of 8.4 ± 0.5 μg/mL. Further purification of the active fraction through successive column chromatography led to the isolation and identification of eleven compounds: three previously undescribed compounds (vernonins X-Z, 1-3) and eight known ones (4-11). The structural characterization of all isolated compounds was conducted using comprehensive 1D and 2D-NMR spectroscopy, as well as high-resolution mass spectrometry (HRMS). The antiplasmodial activity, cytotoxicity, and hemolytic effects of compounds 1-3 and 7-10 were evaluated. Compounds 1, 2, 8, and 9 demonstrated promising antiplasmodial potencies against P. falciparum 3D7, with IC50 values less than 10 μg/mL. However, among these, only compound 8 displayed cytotoxicity against Vero cell lines, with a CC50 of 306.8 ± 1.7 μg/mL (selectivity index [SI] = 39.3). Compounds 1, 2, and 9 did not show any significant cytotoxicity at concentrations up to 1000 μg/mL. Additionally, none of the tested compounds exhibited hemolytic effects on human red blood cells at concentrations up to 100 μg/mL, indicating their safety. These results support the use of V. glabra for malaria treatment and demonstrate that its bioactive metabolites are promising leads in the development of safe and effective antimalarial drugs.

Chlorophyll (Chl) metabolism is pivotal to both photosynthesis and plant senescence and represents one of the most fundamental biological processes on Earth with an estimated annual turnover of 1 billion tons. During Chl degradation, only early catabolites and corresponding enzymes are well characterized, whereas for late-stage degradation products it remains often unclear if their formation involves specific enzymes. Here, we report that the ubiquitous YUCCA10 enzymes from the YUCCA flavin-containing monooxygenase (FMOs) family in land plants, normally implicated in the biosynthesis of indole-3-acetic acid (IAA) as the primary form of auxin, surprisingly catalyze the production of several predominant Chl catabolites via mechanistically distinct Baeyer-Villiger oxidation and subsequent hydrolytic γ-lactam-forming deformylation reactions. These historically postulated but hitherto undiscovered Chl degradation steps on several high molecular weight chl catabolites were verified for YUCCA10 from Vitis vinifera and Coffea arabica, while YUCCA10 from Arabidopsis thaliana lacked this activity. In contrast, all three homologs were able to catalyze the rate-limiting key step in IAA biosynthesis, akin to other YUCCA enzymes. Interestingly, Chl catabolites at physiological concentrations impaired IAA formation by YUCCA10 in vitro, suggesting a key role in leaf senescence through enzymatic feedback regulation of auxin levels.

Abstract:Forgetting and memory formation are 2 distinct processes, both essential for the survival of humans and other animals. The protein Musashi2 (MSI2) was recently discovered as a key regulator of forgetting in the model organism Caenorhabditis elegans, making it an attractive therapeutic target for drug discovery and development. Abstract:Here, we report a fluorescence polarization assay-based screening campaign aimed at identifying MSI2 inhibitors from 2,844 plant extracts. The dichloromethane extract of the aerial parts of Huperzia serrata, a plant traditionally used in Chinese medicine for the treatment of memory dysfunction, was identified as a hit and subsequently subjected to HPLC-based activity profiling. Scale-up isolation identified fatty acids, namely linoleic acid (11: ) and palmitoleic acid (12: ), both eluting within the main active window. As other fractions also seem to display activity, although weaker, their minor constituents were also investigated, revealing 1 new serratane triterpenoid (1: ) and 7 known serratene triterpenoids 2: -8: , their biosynthetic precursor α-onocerin (9: ), as well as an abietane diterpene (10: ). The absolute configuration of compound 1: was established by X-ray crystallography. All isolated compounds were evaluated for their activity, revealing that linoleic acid (11: ) and palmitoleic acid (12: ) inhibited MSI2 with IC50 values of 14.6 and 23.1 µM, respectively, while compound 10: only showed moderate activity (IC50 of 34.4 µM).

The formation of amide bonds is key to the biosynthesis of numerous natural products as well as to the industrial production of pharmaceuticals and valuable chemicals. As an alternative to the often inefficient and wasteful chemical syntheses of amides, emerging sustainable biocatalytic strategies rely on enzymes, such as amide bond synthetases (ABSs). Here we report the characterization of two distinct types of ABSs from the biosynthesis of the typically antibacterial and cytotoxic rubromycin polyketides, hyaluromycin and coumarubrin, which are produced by Streptomyces hyaluromycini and Lentzea tibetensis, respectively. These enzymes, ShRaABS and LtRaABS, are presumed to regiospecifically attach the aminocyclopentenone and aminocoumarin substituents (or their biosynthetic precursors) to the rubromycin backbone. Both ABSs were scrutinized for their substrate tolerance toward amino- and carboxy-donors, establishing LtRaABS as the more promising biocatalyst that allowed the generation of numerous unnatural rubromycins. These compounds include a "Trojan Horse" dopamine congener as the first known rubromycin with antibiotic activity against a Gram-negative Pseudomonas sp. by exploiting the strain's siderophore-uptake machinery. In addition, LtRaABS allowed the azide functionalization of rubromycins, thereby setting the stage for bioorthogonal click chemistry that can be employed in the future, for example, for the generation of antibody-drug conjugates.

The ubiquitous flavoenzymes typically function as oxidoreductases that comprise several distinct main types, including flavoprotein oxidases (FPOs), flavoprotein dehydrogenases (FPDs), and flavoprotein monooxygenases (FPMOs). FPOs and FPDs catalyze two-electron oxidation reactions of organic substrates, typically dehydrogenations, thereby converting oxidized flavin (Flox) into its fully reduced state (Flred). Prior to the next catalytic cycle, molecular oxygen (=dioxygen or O2) or (protein-bound) cofactors facilitate the required Flred reoxidation for FPOs and FPDs. Remarkably, members of these two flavoenzyme types can be homologous with highly similar amino acid compositions and overall structures, as minor protein alterations, particularly in the vicinity of the flavin cofactor, can drastically affect O2 reactivity. Finally, FPMOs incorporate one O2-derived oxygen atom into their substrate. To this end, required electrons for Flred formation and O2 activation either come from NAD(P)H (external FPMOs) or, more rarely, the substrate itself (internal FPMOs). External FPMOs steer O2 reactivity toward the formation of covalent flavin-oxygen adducts primarily at the C4a atom of the flavin's isoalloxazine ring or, in some cases, at the adjacent N5. In contrast, typical internal FPMOs forego the formation of covalent oxygen adducts entirely, although an exception in the form of a flavin-N5-oxide-forming enzyme has been reported. Consequently, natural selection has led to three distinct O2 reactivity patterns in flavoenzymes, which either suppress (FPDs), stimulate (FPOs), or steer (FPMOs) this challenging process. In this review, current knowledge on the relationship between flavoenzymes and O2 is summarized, emphasizing strategies to insert oxygen into organic substrates and counteract uncoupling, while also highlighting open questions and future challenges.

Natural products are an essential source of medicines, accounting for a large proportion of approved drugs nowadays. However, the isolation of active natural products from complex extracts is challenging. To address this bottleneck, a drug discovery strategy was developed in our lab, that combines the screening of an in-house crude plant extract library of more than 2,500 samples with an HPLC-based activity profiling approach. This workflow is used routinely in our group and was successfully applied to numerous natural product drug discovery projects.

The genus Vernonia is one of the largest in the family Asteraceae (Compositae) with almost 3,257 species. It is recognised as a rich source of sesquiterpene lactones (SLs), an important class of bioactive secondary metabolites known for their high structural diversity and significant biological activities. We provide herein a first comprehensive review on the SLs from the genus Vernonia, comprising in addition compounds from closely related plants of the tribe Vernonieae that were only recently reclassified into other genera. Between 1968 and January 2025, chemical investigations of these plants have led to the isolation and characterization of a total of 294 distinct SLs, including 262 naturally occurring compounds and 32 synthetic derivatives, organized into eight main subclasses. These include 125 germacrolides (or germacranolides) comprising 11 synthetic derivatives, 77 hirsutinolides, 34 elemanolides featuring 11 synthetic congeners, 19 guaianolides, 5 vernomargolides, 5 vernogalcanolides, 7 eudesmanolides and 22 dimeric SLs. Additionally, information on the chemistry, distribution, biosynthesis and biological activities (cytotoxic, antiplasmodial, antitrypanosomial, antiinflammatory, antimetastatic and antimicrobial) of these SLs are provided, along with tentative structure–activity relationships for some closely related compounds, which will be valuable for future pharmacological investigations of SLs from present and former members of the genus Vernonia.

Long-chain n-alkane functionalization is relevant for industrial and environmental applications, but it remains a significant challenge due to the inherent stability of their covalent bonds. Biocatalytic approaches offer promising strategies due to their potential for selective, efficient, and environmentally friendly processes. Among the enzyme families known to functionalize long-chain n-alkanes, one iron-binding protein, AlkB, has been characterized. Additionally, two distinct metal-free flavin-dependent enzymes, LadA and AlmA, are presumed to perform this function. Unlike the membrane-bound AlkB, LadA and AlmA are soluble proteins, making them more amenable to engineering and scalable industrial applications. In this study, we attempted to reproduce and optimize the n-alkane monooxygenase activity of LadA. We tested the functionality of this enzyme, an optimized variant, and four novel homologs under in vitro conditions. Despite extensive efforts, we were unable to detect any long-chain n-alkane hydroxylation. Analysis of LadA's protein superfamily and the reported experimental evidence indicate that LadA may be involved in the metabolism of long-chain n-alkanes. However, its role in the first oxo-functionalization step could not be corroborated. Similar conclusions were published regarding AlmA's activity. Altogether, these findings challenge the current understanding of flavoprotein monooxygenases as long-chain n-alkane monooxygenases and underscore the need for further investigation into their biochemical role.

Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015. Since its conception, MIBiG has been regularly updated to expand data coverage and remain up to date with innovations in natural product research. Here, we describe MIBiG version 4.0, an extensive update to the data repository and the underlying data standard. In a massive community annotation effort, 267 contributors performed 8304 edits, creating 557 new entries and modifying 590 existing entries, resulting in a new total of 3059 curated entries in MIBiG. Particular attention was paid to ensuring high data quality, with automated data validation using a newly developed custom submission portal prototype, paired with a novel peer-reviewing model. MIBiG 4.0 also takes steps towards a rolling release model and a broader involvement of the scientific community. MIBiG 4.0 is accessible online at https://mibig.secondarymetabolites.org/. [GRAPHICS] .

Chemical investigations of a methanolic extract of the twigs of Vernonia amygdalina Delile (Asteraceae) resulted in the isolation and identification of three previously undescribed highly oxygenated Delta(7,9(11)) stigmastane-type steroids namely vernonins U-W (1-3) along with six known compounds (4-9). The structural characterization of all the isolated compounds has been conducted via comprehensive 1D and 2D-NMR spectroscopy as well as HRMS. The seven steroidal derivatives 1-7 were evaluated for their antiplasmodial activity against the chloroquine-resistant strain P. falciparum Dd2 (PfDd2) and their hemolytic effect on human red blood cells (RBCs). Vernonins U (1), A (4) and stigmasterol-3-O-beta-d-glucopyranoside (7) showed the highest activity with IC50 values of (5.47 +/- 0.01) mu g/mL, (6.02 +/- 0.13) mu g/mL and (6.34 +/- 0.80) mu g/mL, respectively, against PfDd2, while vernonin W (3) showed moderate activity of (21.20 +/- 0.40) mu g/mL. None of the tested compounds displayed hemolytic effects on human RBCs up to 100 mu g/mL indicating their safety. These results enrich the known chemistry of V. amygdalina and support its use in folk medicine for the treatment of malaria. This encourages further research towards new antiplasmodial drug candidates from this plant.

The non-benzenoid aromatic tropone ring is a structural motif of numerous microbial and plant natural products with potent bioactivities. In bacteria, tropone biosynthesis involves early steps of the widespread CoA-dependent phenylacetic acid (paa) catabolon, from which a shunt product is sequestered and surprisingly further utilized as a universal precursor for structurally and functionally diverse tropone derivatives such as tropodithietic acid or (hydroxy)tropolones. Here, we elucidate the biosynthesis of the antibiotic 3,7-dihydroxytropolone in Actinobacteria by in vitro pathway reconstitution using paa catabolic enzymes as well as dedicated downstream tailoring enzymes, including a thioesterase (TrlF) and two flavoprotein monooxygenases (TrlCD and TrlE). We furthermore mechanistically and structurally characterize the multifunctional key enzyme TrlE, which mediates an unanticipated ipso-substitution involving a hydroxylation and subsequent decarboxylation of the CoA-freed side chain, followed by ring oxidation to afford tropolone. This study showcases a remarkably efficient strategy for 3,7-dihydroxytropolone biosynthesis and illuminates the functions of the involved biosynthetic enzymes.

Steroids are farnesyl diphosphate-derived triterpene derivatives widely distributed in Meliaceae plants that can have several health benefits due to their biological activities. This literature survey on chemical and pharmacological studies of steroids from the Meliaceae plants indicates that 157 distinct steroids classified into six subclasses including (in decreasing number): pregnane-, stigmastane-, ergostane-, cholestane-, androstane- and ecdysterone-type steroids have been reported from a total of 49 plant species. This review aims to provide a reference document compiling information about the occurrence, chemistry and biological activities of meliaceous steroids for the period from 1988 to July 2023. In particular, generalities about the chemistry of steroids with unusual skeletons and underlying biosynthetic pathways are highlighted. In addition, some structural relationships between different compound types and their biological activities are presented. The information used during the writing of this paper was collected from the online libraries PubMed, Google Scholar and Scifinder using the keywords steroids and Meliaceae with no language restriction. This review points out new avenues for further investigations of steroids from plants of the Meliaceae family.

ABSTRACT The genus Pseudomonas is a prolific source of specialized metabolites with significant biological activities, including siderophores, antibiotics, and plant hormones. These molecules play pivotal roles in environmental interactions, influencing pathogenicity, inhibiting microorganisms, responding to nutrient limitation and abiotic challenges, and regulating plant growth. These properties mean that pseudomonads are suitable candidates as biological control agents against plant pathogens. Multiple transposon-based screens have identified a Pseudomonas biosynthetic gene cluster (BGC) associated with potent antibacterial and antifungal activities, which produces 7-hydroxytropolone (7-HT). In this study, we show that this BGC also makes 3,7-dihydroxytropolone (3,7-dHT), which has strong antimicrobial activity toward Streptomyces scabies, a potato pathogen. Through metabolomics and reporter assays, we unveil the involvement of cluster-situated genes in generating phenylacetyl-coenzyme A, a key precursor for tropolone biosynthesis via the phenylacetic acid catabolon. The clustering of these phenylacetic acid genes within tropolone BGCs is unusual in other Gram-negative bacteria. Our findings support the interception of phenylacetic acid catabolism via an enoyl-CoA dehydratase encoded in the BGC, as well as highlighting an essential role for a conserved thioesterase in biosynthesis. Biochemical assays were used to show that this thioesterase functions after a dehydrogenation–epoxidation step catalyzed by a flavoprotein. We use this information to identify diverse uncharacterized BGCs that encode proteins with homology to flavoproteins and thioesterases involved in tropolone biosynthesis. This study provides insights into tropolone biosynthesis in Pseudomonas, laying the foundation for further investigations into the ecological role of tropolone production.IMPORTANCEPseudomonas bacteria produce various potent chemicals that influence interactions in nature, such as metal-binding molecules, antibiotics, or plant hormones. This ability to synthesize bioactive molecules means that Pseudomonas bacteria may be useful as biological control agents to protect plants from agricultural pathogens, as well as a source of antibiotic candidates. We have identified a plant-associated Pseudomonas strain that can produce 3,7-dihydroxytropolone, which has broad biological activity and can inhibit the growth of Streptomyces scabies, a bacterium that causes potato scab. Following the identification of this molecule, we used a combination of genetic, chemical, and biochemical experiments to identify key steps in the production of tropolones in Pseudomonas species. Understanding this biosynthetic process led to the discovery of an array of diverse pathways that we predict will produce new tropolone-like molecules. This work should also help us shed light on the natural function of antibiotics in nature.

Flavoenzymes catalyze numerous redox reactions including the transfer of an O2-derived oxygen atom to organic substrates, while the other one is reduced to water. Investigation of some of these monooxygenases led to a detailed understanding of their catalytic cycle, which involves the flavin-C4α-(hydro)peroxide as hallmark oxygenating species, and newly discovered flavoprotein monooxygenases were generally assumed to operate similarly. However, discoveries in recent years revealed a broader mechanistic versatility, including enzymes that utilize flavin-N5 oxygen adducts for catalysis in the form of the flavin-N5-(hydro)peroxide and the flavin-N5-oxide species. In this review, I will highlight recent developments in that area, including noncanonical flavoenzymes from natural product biosynthesis and sulfur metabolism that provide first insights into the chemical properties of these species. Remarkably, some enzymes may even combine the flavin-N5-peroxide and the flavin-N5-oxide species for consecutive oxygen-transfers to the same substrate and thereby in essence operate as dioxygenases.